Guanosine cyclic 3',5'-phosphate dependent protein kinase, a chimeric protein homologous with two separate protein families

The amino acid sequence of bovine lung cGMP-dependent protein kinase has been determined by degradation and alignment of two primary overlapping sets of peptides generated by cleavage at methionyl or arginyl residues. The protein contains 670 residues in a single N alpha-acetylated chain corresponding to a molecular weight of 76 331. The function of the molecule is considered in six segments of sequence which may correspond to four folding domains. From the amino terminus, the first segment is related to the dimerizing property of the protein. The second and third segments appear to have evolved from an ancestral tandem internal gene duplication, generating twin cGMP-binding domains which are homologous to twin domains in the regulatory subunits of cAMP-dependent protein kinase and to the cAMP-binding domain of the catabolite gene activator of Escherichia coli. The fourth and fifth segments may comprise one domain which is homologous to the catalytic subunits of cAMP-dependent protein kinase, of calcium-dependent phosphorylase b kinase, and of certain oncogenic viral protein tyrosine kinases. The regulatory, amino-terminal half of cGMP-dependent protein kinase appears to be related to a family of smaller proteins that bind cAMP for diverse purposes, whereas the catalytic, carboxyl-terminal half is related to a family of protein kinases of varying specificity and varying sensitivity to regulators. These data suggest that ancestral gene splicing events may have been involved in the fusion of two families of proteins to generate the allosteric character of this chimeric enzyme.     

Amino acid sequence of the regulatory subunit of bovine type I adenosine cyclic 3',5'-phosphate dependent protein kinase

The complete amino acid sequence of the regulatory subunit of type I cAMP-dependent protein kinase from bovine skeletal muscle is presented. The S-carboxymethylated protein was cleaved with cyanogen bromide to provide a complete set of nonoverlapping fragments. These fragments were overlapped and aligned by using peptides generated by proteolytic cleavage. The protein contains 379 amino acid residues corresponding to a molecular weight of 42 804. As in the type II regulatory subunit of cAMP-dependent protein kinase, a pattern of internal gene duplication is observed, which is consistent with two cAMP-binding domains. The two types of regulatory subunit from type I and type II kinase display similarities in domain substructure and in amino acid sequence, which provide a molecular basis for new insight into their regulatory roles. Detailed analyses of the homology of the regulatory subunits of type I and type II cAMP-dependent protein kinase and of similar relationships to cGMP-dependent protein kinase and Escherichia coli catabolite gene activator protein are presented in accompanying reports from this laboratory [Takio, K., Smith, S. B., Krebs, E. G., Walsh, K., & Titani, K. (1984) Biochemistry (second paper of three in this issue); Takio, K., Wade, R. D., Smith, S. B., Krebs, E. G., Walsh, K. A., & Titani, K. (1984) Biochemistry (third paper of three in this issue)].     

Liver isozyme of rabbit glycogen synthase. Amino acid sequences surrounding phosphorylation sites recognized by cyclic AMP-dependent protein kinase

Glycogen synthase, the rate-limiting enzyme in glycogen biosynthesis, has been postulated to exist as isozymes in rabbit liver and muscle (Camici, M., Ahmad, Z., DePaoli-Roach, A. A., and Roach, P. J. (1984) J. Biol. Chem. 259, 2466-2473). Both isozymes share a number of properties including multiple phosphorylation of the enzyme subunit. In the present study, we determined the amino acid sequences surrounding phosphorylation sites in the rabbit liver isozyme recognized by cyclic AMP-dependent protein kinase. Two dominant phosphopeptides (P-1 and P-2) were generated from tryptic digestion. Amino acid sequences of the purified peptides were determined by automated Edman degradation using a gas-phase sequenator. The locations of phosphorylated residues were identified by measuring 32Pi release during Edman degradation cycles. The NH2-terminal sequence of peptide P-1 is S-L-S(P)-V-T-S-L-G-G-L-P-Q-W-E-V-E-E-L-P-V-D-D-L-L-L-P-E-V. This sequence exhibits a strong homology to the site 2 region in the NH2 terminus of the muscle isozyme. The NH2-terminal sequence of peptide P-2 is M-Y-P-R-P-S(P)-S(P)-V-P-P-S-P-L-G-S-Q-A. This sequence shows strong homology to the site 3 region in the COOH terminus of the muscle isozyme. However, some interesting sequence differences were revealed in this region. For example, substitution of serine for alanine at position 6 of peptide P-2 created a new phosphorylation site for cyclic AMP-dependent protein kinase. Phosphorylation of the proline/serine-rich site 3 region correlated with inactivation of the liver isozyme and suggests an important role for this segment of the molecule in the regulation of glycogen synthase. No phosphorylation sites corresponding to sites 1a and 1b of the muscle isozyme were detected. In addition, the results provide definitive chemical proof that glycogen synthase from rabbit liver and muscle are isozymes encoded by distinct messages.     

Chymotryptic inhibitor I from potatoes. The amino acid sequence of subunit A

The amino acid sequence of subunit A of the potato chymotryptic inhibitor I was determined. The sequence was deduced from analysis of fragments and peptides derived from the protein by cleavage with cyanogen bromide, N-bromosuccinimide and dilute acid, and by digestion with trypsin, thermolysin, pepsin and papain. The molecule consists of a single polypeptide chain of 84 residues, which contains two homologous regions each of 13 amino acids. The protein does not appear to be homologous with any other known proteinase inhibitors.     

Sequence comparison of the 63-, 61-, and 59-kDa calmodulin-dependent cyclic nucleotide phosphodiesterases.

Partial protein sequences from the 59-kDa bovine heart and the 63-kDa bovine brain calmodulin-dependent phosphodiesterases (CaM-PDEs) were determined and compared to the sequence of the 61-kDa isozyme reported by Charbonneau et al. [Charbonneau, H., Kumar, S., Novack, J. P., Blumenthal, D. K., Griffin, P. R., Shabanowitz, J., Hunt, D. F., Beavo, J. A. & Walsh, K. A. (1991) Biochemistry (preceding paper in this issue)]. Only a single segment (34 residues) at the N-terminus of the 59-kDa isozyme lacks identity with the 61-kDa isozyme; all other assigned sequence is identical in the two isozymes. Peptides from the 59-kDa isozyme that correspond to residues 23-41 of the 61-kDa protein bind calmodulin with high affinity. The C-terminal halves of these calmodulin-binding peptides are identical to the corresponding 59-kDa sequence; the N-terminal halves differ. The localization of sequence differences within this single segment suggests that the 61- and 59-kDa isozymes are generated from a single gene by tissue-specific alternative RNA splicing. In contrast, partial sequence from the 63-kDa bovine brain CaM-PDE isozyme displays only 67% identity with the 61-kDa isozyme. The differences are dispersed throughout the sequence, suggesting that the 63- and 61-kDa isozymes are encoded by separate but homologous genes.     

Purification of crystallization of transaldolase isozyme I and evidence for different genetic origin of isozymes I and III in Candida utilis

A modified procedure for the purification and crystallization of isozymes I and III of transaldolase from extracts of Candida utilis has been developed which makes both enzymes available in sufficient quantity for structural studies. Each is composed of a pair of identical subunits, but the molecular weight of isozyme I is somewhat larger than that of isozyme III. An important difference is in the number of histidine residues: one per subunit in isozyme III and two per subunit in isozyme I. A nonapeptide containing both histidine residues has now been isolated from isozyme I; its sequence is identical to that of the corresponding segment from isozyme III, except that tyrosine is replaced by histidine: His (in place of Tyr)-Gly-Ile-His-Cys-Asx-Thr-Leu-Leu. This amino acid substitution establishes that two different genes code for the two isozymes.     

Phosphorylation of the alpha subunit of rat brain sodium channels by cAMP-dependent protein kinase at a new site containing Ser686 and Ser687

The alpha subunit of the rat brain sodium channel is phosphorylated by cAMP-dependent protein kinase in vitro and in situ at multiple sites which yield seven tryptic phosphopeptides. Phosphopeptides 1-4 and 7 are derived from phosphorylation sites between residues 554 and 623 in a single large CNBr fragment from the cytoplasmic segment connecting homologous domains I and II of the alpha subunit (Rossie, S., Gordon, D., and Catterall, W. A. (1987) J. Biol. Chem. 262, 17530-17535). In the present work, antibodies were prepared against a synthetic peptide corresponding to residues 676-692 (AbSP15), which contain one additional potential phosphorylation site at Ser686-Ser687 in a different predicted CNBr fragment of this same intracellular segment. AbSP15 recognizes native and denatured sodium channels specifically and immunoprecipitates phosphorylated CNBr fragments of low molecular mass that contain a new site phosphorylated by cAMP-dependent protein kinase. Comparison of tryptic phosphopeptides derived from intact alpha subunits with those derived from the phosphorylated CNBr fragments isolated by immunoprecipitation with AbSP15 indicates that the two previously unidentified phosphopeptides 5 and 6 derived from the intact alpha subunit arise from phosphorylation of the site containing Ser686-Ser687. These results identify a new cAMP-dependent phosphorylation site and show that the major cAMP-dependent phosphorylation sites of the rat brain sodium channel, which are phosphorylated both in vitro and in intact neurons, are all located in a cluster between residues 554 and 687 in the intracellular segment between domains I and II.     

Molecular basis for regulatory subunit diversity in cAMP-dependent protein kinase: crystal structure of the type II beta regulatory subunit

BACKGROUND: Cyclic AMP binding domains possess common structural features yet are diversely coupled to different signaling modules. Each cAMP binding domain receives and transmits a cAMP signal; however, the signaling networks differ even within the same family of regulatory proteins as evidenced by the long-standing biochemical and physiological differences between type I and type II regulatory subunits of cAMP-dependent protein kinase. RESULTS: We report the first type II regulatory subunit crystal structure, which we determined to 2.45 A resolution and refined to an R factor of 0.176 with a free R factor of 0.198. This new structure of the type II beta regulatory subunit of cAMP-dependent protein kinase demonstrates that the relative orientations of the two tandem cAMP binding domains are very different in the type II beta as compared to the type I alpha regulatory subunit. Each structural unit for binding cAMP contains the highly conserved phosphate binding cassette that can be considered the "signature" motif of cAMP binding domains. This motif is coupled to nonconserved regions that link the cAMP signal to diverse structural and functional modules. CONCLUSIONS: Both the diversity and similarity of cAMP binding sites are demonstrated by this new type II regulatory subunit structure. The structure represents an intramolecular paradigm for the cooperative triad that links two cAMP binding sites through a domain interface to the catalytic subunit of cAMP-dependent protein kinase. The domain interface surface is created by the binding of only one cAMP molecule and is enabled by amino acid sequence variability within the peptide chain that tethers the two domains together.     

Amino acid sequence of the N-terminal non-collagenous segment of dermatosparactic sheep procollagen type I.

The non-collagenous N-terminal segment of type I procollagen from dermatosparactic sheep skin was isolated in the form of the peptide Col 1 from a collagenase digest of the protein. The peptide has a blocked N-terminus, which was identified as pyrrolid-2-one-5-carboxylic acid. Appropriate overlapping fragments were prepared from reduced and alkylated peptide Col 1 by cleavage with trypsin at lysine, arginine and S-aminoethyl-cysteine residues and by cleavage with staphylococcal proteinase at glutamate residues. Amino acid sequence analysis of these fragments by Edman degradation and mass spectrometry established the whole sequence of peptide Col 1 except for a peptide junction (7--8) and a single Asx residue (44), and demonstrated that peptide Col 1 consists of 98 amino acid residues. The N-terminal portion of peptide Col 1 (86 residues) shows an irregular distribution of glycine, whereas the C-terminal portion (12 residues) possesses the triplet structure Gly-Xy and is apparently derived from the precursor-specific collagenous domain of procollagen. The central region of the peptide contains ten cysteine residues located between positions 18 and 73 and shows alternating polar and hydrophobic sequence elements. The regions adjacent to the cysteine-rich portion have a hydrophilic nature and are abundant in glutamic acid. The data are consistent with previous physicochemical and immunological evidence that distinct regions at the N- and C-termini of the non-collagenous domain possess a less rigid conformation than does the central portion of the molecule.     

Characterization of the antigenic structure of herpes simplex virus type 1 glycoprotein C through DNA sequence analysis of monoclonal antibody-resistant mutants.

Earlier studies of a group of monoclonal antibody-resistant (mar) mutants of herpes simplex virus type 1 glycoprotein C (gC) operationally defined two distinct antigenic sites on this molecule, each consisting of numerous overlapping epitopes. In this report, we further define epitopes of gC by sequence analysis of the mar mutant gC genes. In 18 mar mutants studied, the mar phenotype was associated with a single nucleotide substitution and a single predicted amino acid change. The mutations were localized to two regions within the coding sequence of the external domain of gC and correlated with the two previously defined antigenic sites. The predicted amino acid substitutions of site I mutants resided between residues Gln-307 and Pro-373, whereas those of site II mutants occurred between amino acids Arg-129 and Glu-247. Of the 12 site II mutations, 9 induced amino acid substitutions within an arginine-rich segment of 8 amino acids extending from residues 143 to 151. The clustering of the majority of substituted residues suggests that they contribute to the structure of the affected sites. Moreover, the patterns of substitutions which affected recognition by antibodies with similar epitope specificities provided evidence that epitope structures are physically linked and overlap within antigenic sites. Of the nine epitopes defined on the basis of mutations, three were located within site I and six were located within site II. Substituted residues affecting the site I epitopes did not overlap substituted residues of site II, supporting our earlier conclusion that sites I and II reside in spatially distinct antigenic domains. A computer analysis of the distribution of charged residues and the predicted secondary structural features of wild-type gC revealed that the two antigenic sites reside within the most hydrophilic regions of the molecule and that the antigenic residues are likely to be organized as beta sheets which loop out from the surface of the molecule. Together, these data and our previous studies support the conclusion that the mar mutations identified by sequence analysis very likely occur within or near the epitope structures themselves. Thus, two highly antigenic regions of gC have now been physically and genetically mapped to well-defined domains of the protein molecule.     

Amino acid sequence of alcohol dehydrogenase from the thermophilic bacterium Thermoanaerobium brockii

The complete amino acid sequence of alcohol dehydrogenase of Thermoanaerobium brockii (TBAD) is presented. The S-carboxymethylated protein was cleaved at methionine residues (with cyanogen bromide) to provide a set of 10 nonoverlapping fragments accounting for 90% of the sequence. These fragments were then overlapped and aligned, and the sequence was completed by using peptides generated by proteolytic cleavage at lysine residues (with Achromobacter protease I). The protein subunit contained 352 amino acid residues corresponding to a molecular weight of 37,652. The sequence showed about 35% identity with that of the prokaryotic Alcaligenes eutrophus alcohol dehydrogenase and about 25% identity with any one of the eukaryotic alcohol/polyol dehydrogenases known today. Of these, only 18 residues (5%) are strictly conserved: 11 Gly, 2 Asp, and 1 each of Cys, His, Glu, Pro, and Val.     

Molecular cloning and targeted mutagenesis of the gene psaF encoding subunit III of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803

Photosystem I is one of the two multisubunit pigment-protein complexes in the thylakoid membranes of cyanobacteria. Subunit III of photosystem I complex was isolated from a mutant of the cyanonbacterium Synechocystis sp PCC 6803, which lacks subunit II. The sequence of its NH2-terminal residues was determined and corresponding oligonucleotide probes were used to isolate the gene encoding this subunit. The gene, designated as psaF, codes for a mature protein of 15705 Da that is synthesized with a 23-amino acid extension. The deduced amino acid sequence is homologous to subunit III from spinach and Chlamydomonas reinhardtii. The presequence of subunit III shows characteristics typical of bacterial presequences and exhibits remarkable amino acid identity around the proteolytic processing site when compared to corresponding regions from the precursors of eukaryotic subunit III. There are two conserved hydrophobic regions in the mature subunit III which may cross or interact with thylakoid membrane. The gene psaF exists as a single copy in the genome and is expressed as a monocistronic RNA. A stable mutant strain in which the gene psaF was replaced by a gene conferring resistance to kanamycin was generated by targeted mutagenesis. Photoautotrophic growth of the mutant strain was comparable with that of the wild type suggesting that function of subunit III is dispensable for photosynthesis in Synechocystis sp. PCC 6803. Addition of more MgSO4 to BG11 medium enhanced growth of the mutant strain but not of the wild type cells.     

Monoclonal antibodies as structural probes of surface residues in the regulatory subunit of cAMP-dependent protein kinase II from porcine heart

Two murine monoclonal antibodies (H5 and B6) generated against bovine heart type II regulatory subunit of cAMP-dependent protein kinase were shown to cross-react equally well with the homologous subunit from porcine heart. The antibodies demonstrated specificity for only the type II regulatory subunit and showed negligible cross-reactivity with the type I regulatory subunit, the catalytic subunit, and cGMP-dependent protein kinase. Following limited proteolysis of type II regulatory subunit with chymotrypsin, the H5 monoclonal antibody was shown to cross-react with the Mr = 37,000 cAMP-binding domain corresponding to the COOH-terminal region of the polypeptide chain. To more specifically localize the antigenic sites, the porcine type II regulatory subunit was carboxymethylated and cleaved with cyanogen bromide. Both monoclonal antibodies cross-reacted with the NH2-terminal CNBr peptide, and this peptide demonstrated affinities similar to native bovine type II regulatory subunit in competitive displacement radioimmunoassays. Tryptic cleavage of this CNBr fragment destroyed all antigenicity for both monoclonal antibodies, whereas antigenicity was retained following chymotryptic digestion. A single major immunoreactive chymotryptic fragment that cross-reacted with H5 was isolated by gel filtration and reverse phase high performance liquid chromatography. this peptide retained the complete antigenic site and had the following sequence: Asn-Pro-Asp-Glu-Glu-Glu-Glu-Asp-Thr-Asp-Pro-Arg-Val-Ile-His-Pro-Lys-Thr-Asp-Gl n. This antigenic site was localized just beyond the major site of autophosphorylation, approximately a third of the distance from the NH2-terminal end of the polypeptide chain.     

Crotalus adamanteus phospholipase A2-α: Subunit structure,NH2-terminal sequence,and homology with other phospholipases

Automated Edman degradation of reduced and carboxymethylated phospholipase A₂-α from Crotalus adamanteus venom revealed a single amino acid sequence extending 30 residues into the protein from the amino terminus. The singularity of the sequence and the yields of the phenylthiohydantoin amino acids thus obtained indicate that the subunits comprising the phospholipase dimer are identical. Further chemical evidence in support of subunit identity was obtained by cleavage of phospholipase A₂-α with cyanogen bromide. Compositional analysis of the protein revealed one residue of methionine per monomer and the sequence determination placed this amino acid at position 10 in the sequence of 133 amino acids. Cyanogen bromide cleavage of the protein, followed by reduction and carboxymethylation afforded the expected 2 fragments: an NH₂-terminal decapeptide (CNBr-1) and a larger COOH-terminal fragment of 123 residues (CNBr-II). Automated Edman degradation of the latter has extended the sequence analysis to 54 residues in the NH₂-terminal segment of the monomer chain. Comparison of this sequence with those derived for phospholipases from other snake venoms, from bee venom, and from porcine pancreas has revealed striking homologies in this region of the molecules. As expected on the basis of their phylogenetic classification, the phospholipases from the pit vipers C. adamanteus and Agkistrodon halys blomhoffii are more similar to one another in sequence than to the enzyme from the more distantly related viper, Bitis gabonica. Furthermore, the very close similarities in sequence observed among all of these phospholipases in regions corresponding to residues 24 through 53 in the C. adamanteus enzyme suggest that this segment of the polypeptide plays an important role in phospholipase function and probably constitutes part of the active site.     

Structural organization of aldehyde dehydrogenases probed by limited proteolysis

Incubation of cytosolic and mitochondrial aldehyde dehydrogenases with trypsin or Glu-C protease under native conditions causes a time-dependent loss of dehydrogenase activity and the production of protein fragments. For evaluation of the results, termination of the reactions with a specific protease inhibitor is especially important in the case of the Glu-C protease. Cleavage site determination by SDS/polyacrylamide gel electrophoresis and sequence analysis identified protease-sensitive amino acid residues at two internal regions spanning positions 248-268 (region 1) and 397-399 (region 2) and at positions in the N-terminal segment (region 3). Region 1 encompasses several cleavages and is sensitive to both proteases in both aldehyde dehydrogenases. Further, it is in a conserved segment and correlates with reactive residues and regions ascribed functional roles. It also correlates with exon borders in the corresponding genes. Combined, the results define region 1 as an important and highly accessible segment of the protein. Region 2 is also adjacent to a conserved segment but lacks further correlation with special properties and appears just to represent an accessible region. The internally cleaved subunits retain a tetrameric configuration as calculated from exclusion chromatography and polyacrylamide gel electrophoresis under native conditions, suggesting that the quaternary structure is not dependent on covalently linked domains within the subunits. Furthermore, the fragments can bind to AMP-Sepharose, suggesting that some functional properties are retained within the cleaved tetramers. However, cleavage at position 35 appears to cause a large fragment (36-263) to be released from the tetramer, suggesting a role of an N-terminal segment or arm (at or before region 3) in subunit interactions.     

Signal and membrane anchor functions overlap in the type II membrane protein I gamma CAT

I gamma CAT is a hybrid protein that inserts into the membrane of the endoplasmic reticulum as a type II membrane protein. These proteins span the membrane once and expose the NH2-terminal end on the cytoplasmic side and the COOH terminus on the exoplasmic side. I gamma CAT has a single hydrophobic segment of 30 amino acid residues that functions as a signal for membrane insertion and anchoring. The signal-anchor region in I gamma CAT was analyzed by deletion mutagenesis from its COOH-terminal end (delta C mutants). The results show that the 13 amino acid residues on the amino-terminal side of the hydrophobic segment are not sufficient for membrane insertion and translocation. Mutant proteins with at least 16 of the hydrophobic residues are inserted into the membrane, glycosylated, and partially proteolytically processed by a microsomal protease (signal peptidase). The degree of processing varies between different delta C mutants. Mutant proteins retaining 20 or more of the hydrophobic amino acid residues can span the membrane like the parent I gamma CAT protein and are not proteolytically processed. Our data suggest that in the type II membrane protein I gamma CAT, the signals for membrane insertion and anchoring are overlapping and that hydrophilic amino acid residues at the COOH-terminal end of the hydrophobic segment can influence cleavage by signal peptidase. From this and previous work, we conclude that the function of the signal-anchor sequence in I gamma CAT is determined by three segments: a positively charged NH2 terminus, a hydrophobic core of at least 16 amino acid residues, and the COOH-terminal flanking hydrophilic segment.     

Phosphorylation of the 48-kDa subunit of the glycine receptor by protein kinase C.

The postsynaptic glycine receptor purified from rat spinal cord is rapidly and specifically phosphorylated by protein kinase C. The target for phosphorylation is the strychnine-binding subunit of the receptor (molecular mass of approximately 48 kDa), which is phosphorylated on serine residues to a final stoichiometry of approximately 0.8 mol of phosphate/mol of subunit. The 48-kDa phosphoprotein was analyzed by proteolytic cleavage and peptide mapping in order to localize the site of phosphorylation within the receptor molecule. Examination of the 32P-labeled receptor fragments generated by digestion with N-chlorosuccinimide, cyanogen bromide, and endoproteinase lysine C and of the deduced amino acid sequence of the 48-kDa protein (Grenningloh, G., Rienitz, A., Schmitt, B., Methfessel, C., Zensen, M., Beyreuther, K., Gundelfinger, E. D., and Betz, H. (1987) Nature 328, 215-220) indicates that the phosphorylation site is located in a region corresponding to the major intracellular loop of the predicted structure of the glycine receptor subunit and suggests serine 391 as the phosphorylated residue. In fact, a synthetic peptide corresponding to residues 384-392 of the 48-kDa subunit was specifically phosphorylated by protein kinase C. Moreover, tryptic digests of this phosphopeptide and of the phosphorylated 48-kDa subunit of the glycine receptor migrated to the same position in two-dimensional peptide mapping. Furthermore, antibodies elicited against peptide 384-392 were shown to inhibit the protein kinase C-dependent phosphorylation of the 48-kDa polypeptide. Interestingly, the relative position of the phosphorylated domain is similar to those known or proposed to be phosphorylated in other ligand-gated ion channel receptor subunits, thus suggesting further the existence of a homologous regulatory region in these receptor proteins.     

The gamma-subunit of skeletal muscle phosphorylase kinase contains two noncontiguous domains that act in concert to bind calmodulin

Phosphorylase kinase is a Ca2+-regulated, multisubunit enzyme that contains calmodulin as an integral subunit (termed the delta-subunit). Ca2+-dependent activity of the enzyme is thought to be regulated by direct interaction of the delta-subunit with the catalytic subunit (the gamma-subunit) in the holoenzyme complex. In order to systematically search for putative calmodulin (delta-subunit)-binding domain(s) in the gamma-subunit of phosphorylase kinase, a series of 18 overlapping peptides corresponding to the C terminus of the gamma-subunit was chemically synthesized using a tea bag method. The calmodulin-binding activity of each peptide was tested for its ability to inhibit Ca2+/calmodulin-dependent activation of myosin light chain kinase. Data were obtained indicating that two distinct regions in the gamma-subunit, one spanning residues 287-331 (termed domain-N) and the other residues 332-371 (domain-C), are capable of binding calmodulin with nanomolar affinity. Peptides from both of these two domains also inhibited calmodulin-dependent reactivation of denatured gamma-subunit. The interactions of peptides from both domain-N and domain-C with calmodulin were found to be Ca2+-dependent. Dixon plots obtained using mixtures of peptides from domain-N and domain-C indicate that these two domains can bind simultaneously to a single molecule of calmodulin. Multiple contacts between the gamma-subunit and calmodulin (delta-subunit), as indicated by our data, may help to explain why strongly denaturing conditions are required to dissociate these two subunits, whereas complexes of calmodulin with most other target enzymes can be readily dissociated by merely lowering Ca2+ to submicromolar concentrations. Comparison of the sequences of the two calmodulin-binding domains in the gamma-subunit of phosphorylase kinase with corresponding regions in troponin I indicates similarities that may have functional and evolutionary significance.     

A comparison of the cyclic nucleotide-dependent protein kinases using chemical cleavage at tryptophan and cysteine

cGMP-dependent protein kinase (G-kinase) and the regulatory subunit of type I (RI) cAMP-dependent protein kinase (A-kinase) both contain a phosphorylation site located near the NH2 terminus of each enzyme. These sites can be utilized as convenient markers for the determination of the position of an amino acid residue susceptible to either chemical or enzymatic digestion. Using the tryptophan-specific reagent, N-chlorosuccinimide, the approximate location along the polypeptide chain of six reactive tryptophans in G-kinase and three reactive residues in RI were identified. Similarly, cleavage with cyanide was used to locate free and disulfide-bonded cysteines in both proteins. The approximate positions of nine cysteines in G-kinase were determined along with the location of the interchain disulfide bond and an intrachain disulfide bond. RI was found to contain three cyanide-reactive cysteines, two of which are involved in interchain disulfide bonding. A comparison of the positions of the cysteines and tryptophans determined by chemical cleavage in G-kinase and RI, with the positions of cysteine and tryptophan in the known sequence of the type II A-kinase, support the structural relationships between these enzymes. Comparison with subsequently reported primary sequences of all three enzymes indicates the limits of precision of this chemical cleavage procedure.